Mycobacterium Antigens

ABSTRACT

There is provided a diagnostic reagent for use in the detection of  M. bovis  or  M. tuberculosis  infection in an animal, comprising a peptide which has an epitope from  Mycobacterium bovis  hypothetic protein Mb3645c (SEQ ID NO: 1) or an epitope from a polypeptide having at least 76% identity with SEQ ID NO: 1.

FIELD OF INVENTION

The present invention relates to antigens for use in the detection ofmycobacterium infections, particularly Mycobacterium tuberculosis and M.bovis, in mammals such as cattle.

BACKGROUND OF THE INVENTION

M. tuberculosis and M. bovis are important pathogens of man and animals.M. tuberculosis is thought to infect up to a third of the world's humanpopulation, remaining undetected during a latent phase of infection andreactivating to cause 10 million cases of tuberculosis and otherdiseases per year resulting in 2 million deaths (Corbett et al., 2003).M. bovis, which has more than 99.9% sequence identity with M.tuberculosis, is the causative agent of bovine tuberculosis (BTB) andalso causes disease in human. BTB represents a significant economicburden to the agricultural industries of various countries including theUnited Kingdom (Krebs, 1997; DEFRA, 2006).

Current methods of control for these mycobacterial infections centre onthe live attenuated vaccine M. bovis bacillus Calmette-Guerin (BCG) anddiagnosis using an intradermal skin test with a purified proteinderivative (PPD, tuberculin) harvested from mycobacterial cultures. ThePPD skin test relies on a cellular immune response which is mounted incattle with a mycobacterial infection. BTB control measures as appliedfor example in the United Kingdom and other European countries comprisea “test and slaughter” strategy where a positive result to a routineskin test with the single intradermal comparative tuberculin test(SICTT), leads to mandatory slaughter. In human populations the BCGvaccine has been used. However, BCG vaccination programs are hampered bywidely differing rates of protection in different populations withefficacies that range from 0 to 80% (Colditz et al., 1994; Fine, 1995).In addition, vaccination sensitises individuals to tuberculin therebyinterfering with diagnosis.

In addition to BTB skin tests, blood-based diagnostic assays thatmeasure antigen-induced lymphokine production such as the interferongamma (IFN-γ) are also under consideration. The cytokine IFN-γ appearsto be critical in the development of immunity to M. tuberculosis. Forexample, both mice with a disrupted IFN-γ gene and humans with mutatedIFN-γ receptor are highly susceptible to mycobacterial infections.However, specificity constraints are associated with the use of PPD insuch assays. These arise due to the crude mixture of M. bovis proteinsthat PPD contains, many of which are cross-reactive with the BCG vaccinestrain and environmental mycobacterial species such as M. avium and M.intracellulare.

Previous studies have demonstrated that diagnostic reagents whichdistinguish between vaccinated and infected cattle can be developedusing specific, defined antigens that are present in virulent M. bovisbut absent from the BCG. Genetic analysis of BCG has revealed thatseveral large genomic regions have been deleted during attenuation andsubsequent prolonged propagation in culture. These regions have beencharacterised, and antigens from one of these regions, RD1, have beenstudied extensively in several species including humans and cattle. Forexample, it has been demonstrated that protein or peptide cocktailscomposed of two RD1 region antigens, ESAT-6 and CFP-10, can be used todistinguish between M. bovis infected and BCG-vaccinated cattle. TheESAT-6/CFP-10 assay is reported to have a sensitivity of at least 77.9%in cattle with confirmed tuberculosis, and a specificity of 100% inBCG-vaccinated and non-vaccinated cattle (Vordermeier et al. 2001).

However, the level of sensitivity achieved with these antigens has notreached that of tuberculin. It would, therefore, be desirable to provideother antigens in order to achieve this desired sensitivity. The presentinvention accordingly addresses the problem of providing furtherdiscriminatory diagnostic reagents for the detection of mycobacterialinfections.

Camus et al. (Microbiology (2002) 148 2967-2973) and the associated NCBIAccession no. NP_218132 is a disclosure of the genome sequence of M.tuberculosis H37Rv, including the gene encoding Rv3615c. There is nosuggestion of the use of the Rv3615c polypeptide or portions of itwithin a reagent for use in detection of M. bovis or M. tuberculosisinfection in an animal.

Gamier et al. (Proc. Natl. Acad. Sci. U.S.A. (2003) 100 7877-7882 andthe associated NCBI Accession no. NP_857284 is a disclosure of thegenome sequence of M. bovis, including the gene encoding Mb3645c. Thereis no suggestion of the use of the Mb3645c polypeptide or portions of itwithin a reagent for use in detection of M. bovis or M. tuberculosisinfection in an animal.

US2003/0129601 discloses a comparison of the genome sequences of M.tuberculosis and M. leprae and reports a total of 644 common proteinsequences. It is proposed that these sequence may have a variety of usesincluding potential as drug targets, diagnostic antigens or subunitvaccine compositions. The inventors for the present application havefound that one of the sequences has particular efficacy in the diagnosisof M. bovis or M. tuberculosis infection.

SUMMARY OF INVENTION

According to the present invention there is provided a diagnosticreagent, in particular for use in the detection of M. bovis or M.tuberculosis infection in an animal, comprising a peptide which has anepitope from M. bovis hypothetic protein Mb3645c (SEQ ID NO: 1) or anepitope from a polypeptide having at least 76% identity with SEQ IDNO: 1. The animal may be a mammal and preferably is a human being or abovine species, for example a domestic cow. Alternatively, the mammalmay be a badger. In a further alternative, the animal may be a fish or abird species. The detection may take place by analysis of a sampleobtained from the animal, such as a blood, saliva, faecal or tissuesample.

M. bovis hypothetical protein Mb3645c has the amino acid sequence:

(SEQ ID NO: 1) MTENLTVQPE RLGVLASHHD NAAVDASSGV EAAAGLGESVAITHGPYCSQ FNDTLNVYLT AHNALGSSLH TAGVDLAKSL RIAAKIYSEA DEAWRKAIDG LFT.

Mb3645c is the M. bovis equivalent of M. tuberculosis Rv3615c, which hasan identical amino acid sequence. References herein to Mb3645c are,therefore, to be taken as including a reference to Rv3615c, unlessotherwise implied or specified.

“Detection of infection” as mentioned above indicates that an animalwhich is infected with M. bovis or M. tuberculosis can be detected and,for example, may be distinguished from an animal which has beenvaccinated against infection by one or both of these bacteria, forexample, by use of the BCG vaccine. The ability to distinguish betweenthese states using a peptide having an epitope from Mb3645c issurprising, in view of the presence of the nucleic acid sequenceencoding this protein in all of M. bovis, M. tuberculosis and the liveattenuated vaccine BCG.

As described below, it has surprisingly been found that the knownhypothetical protein Mb3645c comprises an epitope which can be used fordiagnostic purposes, for example in the specific recognition of an M.bovis—or M. tuberculosis—infected mammal. This is because of theinventors' unexpected discovery that, as mentioned above, although thegene encoding Mb3645c is present in all of M. bovis, M. tuberculosis andthe live attenuated vaccine BCG, exposure of an animal or a sample froman animal to an epitope from Mb3645c only causes a detectable immuneresponse in an animal infected with M. bovis or M. tuberculosis (or in asample from such a animal). Such a response is not detectable in anuninfected animal (or a sample from one), even when that animal has beenadministered the BCG vaccine.

Based on an NCBI protein BLAST search, the closest known protein toMb3645c (other than Rv3615c) is a hypothetical protein MAP3219c from M.avium which shares 75% sequence identity with Mb3645c. The presentinvention excludes any epitope in MAP3219c which is not also found inMb3645c.

As used herein, the term “epitope” refers to the amino acids (typicallya group of around 5 or more amino acids) within a peptide sequence whichare essential in the generation of an immune response and which can,therefore, be used in a diagnostic test. The immune response may be anantibody mediated immune response, but may also be a non-antibodymediated immune response, for example, an immune response which can bedetected by means of a cell-mediated immunity (CMI) assay. Therefore,the epitope may be one which is recognisable by a T cell, for example bybinding of a T cell receptor to the epitope.

The epitope may comprise consecutive amino acids, or the amino acidsforming the epitope may be spaced apart from one another. In the lattercase, the nature of the amino acids between the amino acids forming theepitope may not be crucial to the activity and may be varied, providedthat the tertiary structure of the epitope is maintained, for example sothat an immune response such as a cell-mediated immune response canoccur in response to the presence of the epitope. Determination of theamino acids which form an epitope or part of an epitope can beundertaken using routine methods. For example, one of a series of smallmutations such as point mutations may be made to a peptide and themutated peptide assayed to determine whether the immunogenic ordiagnostic activity has been retained. Where it has, then the variantretains the epitope. If activity has been lost, then the mutation hasdisrupted the epitope and so must be reversed.

Suitably, the diagnostic peptide has less than 103 amino acids, forexample up to 100 amino acids, for example up to 75 amino acids, forexample up to 50 amino acids, for example up to 25 amino acids, forexample up to 20 amino acids. It may comprise a truncated form ofMb3645c.

The diagnostic reagent peptide may comprise a series of consecutiveamino acids from within SEQ ID NO: 1 or from within a polypeptide havingat least 76% identity with SEQ ID NO: 1.

The diagnostic reagent peptide may comprise an epitope from (i.e.,contained in) one or more of the group of peptides consisting of SEQ IDNOs 2-13, which are defined as follows:

(SEQ ID NO: 2) MTENLTVQPE RLGVLASHHD; (SEQ ID NO: 3)PERLGVLASH HDNAAVDASS; (SEQ ID NO: 4) SHHDNAAVDA SSGVEAAAGL;(SEQ ID NO: 5) DASSGVEAAA GLGESVAITH; (SEQ ID NO: 6)AAGLGESVAI THGPYCSQFN; (SEQ ID NO: 7) AITHGPYCSQ FNDTLNVYLT;(SEQ ID NO: 8) SQFNDTLNVY LTAHNALGSS; (SEQ ID NO: 9)VYLTAHNALG SSLHTAGVDL; (SEQ ID NO: 10) LGSSLHTAGV DLAKSLRIAA;(SEQ ID NO: 11) GVDLAKSLRI AAKIYSEADE; (SEQ ID NO: 12)RIAAKIYSEA DEAWRKAIDG;  and (SEQ ID NO: 13) AKIYSEADEA WRKAIDGLFT.

The peptides of SEQ ID NO: 2-13 are overlapping 20-mer peptides whichencompass the complete Mb3645c sequence of SEQ ID NO: 1. As demonstratedbelow, these peptides comprise epitopes which can be used for diagnosticpurposes, for example in the specific recognition of an M. bovis—or M.tuberculosis—infected mammal.

In one embodiment of the invention, the diagnostic reagent comprises oneor more peptides each selected from the group of peptides consisting ofSEQ ID NOs: 1-13. The diagnostic reagent may comprise at least two,three, four, five, six, seven, eight, nine, ten or more peptides with anepitope from one or more peptides each selected from the group ofpeptides consisting of SEQ ID NOs: 1-13. For example, the diagnosticreagent may comprise at least two, three, four, five, six, seven, eight,nine, ten or more peptides each defined by any of SEQ ID NOs: 1-13.

In another embodiment, the diagnostic reagent comprises a peptide havingan epitope from the peptide of SEQ ID NO: 12 and/or 13. This diagnosticreagent may for example comprise the peptide of SEQ ID NO: 12 and/or 13.

In a further embodiment, the diagnostic reagent comprises a peptidehaving one or more epitopes from one or more of the group of peptidesconsisting of SEQ ID NOs: 7, 8, 9, 10, 11, 12 and 13, or the groupconsisting of SEQ ID NOs: 9, 10, 11, 12 and 13 or the group consistingof SEQ ID NOs: 7, 8, 9, 10, 12 and 13. This diagnostic reagent may forexample comprise any one or more peptides each selected from the groupof peptides consisting of SEQ ID NOs: 9, 10, 11, 12 and 13. In anotherembodiment, the diagnostic reagent comprises a peptide having one ormore epitopes from one or more of the group of peptides consisting ofSEQ ID NOs: 2 and 9-13.

The peptides of SEQ ID NOs: 1 and 8, 9, 10, 11, 12 and 13 (especially10, 11, 12 and 13) contain dominant epitopes recognised by bovine Tcells and are therefore particularly useful in the diagnostic reagent ofthe invention.

The diagnostic reagent may, for example, comprise a combination ofepitopes derived from any one or more of the groups of peptides set outbelow:

SEQ ID NOs SEQ ID NOs SEQ ID NOs SEQ ID NOs SEQ ID NOs 2, 9 2, 10 2, 112, 12 2, 13 2, 9, 10 2, 9, 11 2, 9, 12 2, 9, 13 2, 10, 11 2, 10, 12 2,10, 13 2, 11, 12 2, 11, 13 2, 12, 13 2, 9, 10, 11 2, 9, 10, 12 2, 9, 10,13 2, 10, 11, 12 2, 10, 11, 13 2, 11, 12, 2, 9, 10, 11, 2, 9, 10, 11, 2,9, 10, 12, 9, 10 13 12 13 13 9, 11 9, 12 9, 13 9, 10, 11 9, 10, 12 9,10, 13 9, 10, 11, 12 9, 10, 11, 13 9, 10, 12, 13 10, 11 10, 12 10, 1310, 11, 12 10, 11, 13 10, 12, 13 11, 12 11, 13 11, 12, 13 12, 13 10, 12,13 8, 9 8, 10 8, 12 8, 13 8, 9, 10 8, 10, 12 8, 12, 13 8, 9, 12 8, 9, 138, 10, 12 8, 10, 13 7, 8, 10, 12 8, 10, 12, 13 9, 10 9, 12 9, 13 9, 10,12 9, 12, 13

The diagnostic reagent may thus comprise any combination of peptidesselected from those listed above, or any combination of the listedcombinations.

Alternatively, the diagnostic reagent may comprise peptides having allof the epitopes from the group of peptides consisting of, for example,SEQ ID NOs: 12-13, or consisting of SEQ ID NOs: 11-13, or consisting ofSEQ ID NOs: 10-13, or consisting of SEQ ID NOs: 9-13, or consisting ofSEQ ID NOs: 8-13, or consisting of SEQ ID NOs: 7-13, or consisting ofSEQ ID NOs: 2-13. For example, the diagnostic reagent may comprise allof the peptides from the group of peptides consisting of, for example,SEQ ID NOs: 12-13, or consisting of SEQ ID NOs: 11-13, or consisting ofSEQ ID NOs: 10-13, or consisting of SEQ ID NOs: 9-13, or consisting ofSEQ ID NOs: 8-13, or consisting of SEQ ID NOs: 7-13, or consisting ofSEQ ID NOs: 2-13.

The diagnostic reagent may also comprise a fusion peptide in whichfragments derived from SEQ ID NO: 1 or a polypeptide having at least 76%identity thereto have been joined.

The diagnostic reagent Mb3645c-based peptides as defined herein may beused on their own or with one or more other peptides, for example toachieve greater sensitivity and/or specificity of a diagnostic test. Forexample, the diagnostic reagent may in addition comprise one or morepolypeptides or peptides derived from ESAT-6 (SEQ ID NO: 14) and/or theCFP-10 (SEQ ID NO: 15) polypeptides, in which ESAT-6 has the amino acidsequence:

(SEQ ID NO: 14) MTEQQWNFAG IEAAASAIQG NVTSIHSLLD EGKQSLTKLAAAWGGSGSEA YQGVQQKWDA TATELNNALQ NLARTISEAG QAMASTEGNV TGMFA;

and in which CFP-10 has the amino acid sequence:

(SEQ ID NO: 15) MAEMKTDAAT LAQEAGNFER ISGDLKTQID QVESTAGSLQGQWRGAAGTA AQAAVVRFQE AANKQKQELD EISTNIRQAG VQYSRADEEQ QQALSSQMGF.

For example, the peptides derived from ESAT-6 may be the peptides of SEQID NO: 16-21, which are:

(SEQ ID NO: 16) MTEQQWNFAG IEAAAS; (SEQ ID NO: 17) AGIEAAASAI QGNVTS;(SEQ ID NO: 18) AIQGNVTSIH SLLDEG; (SEQ ID NO: 19) KWDATATELN NALQNL; and (SEQ ID NO: 20) GQAMASTEGN VTGMFA.

The peptides derived from CFP-10 may be the peptides of SEQ ID NOs21-25, which are:

(SEQ ID NO: 21) MAEMKTDAAT LAQEAGNF; (SEQ ID NO: 22) QEAGNFERIS GDLKTQ;(SEQ ID NO: 23) VVRFQEAANK QKQELDEI; (SEQ ID NO: 24)NIRQAGVQYS RADEEQQQ;  and (SEQ ID NO: 25) RADEEQQQAL SSQMGF.

The ESAT-6 and CFP-10 peptides of SEQ ID NOs 16-25 have been disclosedin Vordemeier et al. (2001) and provide a useful diagnostic fordetection of M. bovis—and/or M. tuberculosis—infected animals. Used incombination with the Mb3645c-derived peptides, as defined here, a moresensitive diagnostic reagent is obtained.

The diagnostic reagent according to present invention may accordingly bespecific for M. bovis and/or M. tuberculosis.

The diagnostic reagent may be used in the detection of an M.bovis—and/or M. tuberculosis—infected mammal, for example an M.bovis-infected cow.

Also provided according to the present invention is a diagnostic kitcomprising a diagnostic reagent as defined herein. The diagnosticreagent may, in particular, be able to detect an M. bovis—or M.tuberculosis—infected mammal. Preferably, the diagnostic reagent is ableto differentiate between an M. bovis—and/or M. tuberculosis—infectedmammal and a mammal vaccinated against M. bovis or M. tuberculosis (forexample, a mammal vaccinated with the live attenuated vaccine BCG).

The diagnostic kit may be of particular use in the detection of a M.bovis—and/or M. tuberculosis—infected mammal which is not susceptible todiagnosis by the ESAT-6/CFP-10 assay as described in Vordemeier et al.(2001).

The diagnostic kit may comprise one or more peptides each selected fromthose having amino acid sequences of SEQ ID NOs 1-13 and optionallyadditionally comprise one or more peptides each selected from thosehaving amino acid sequences of SEQ ID NOs 16-25.

The diagnostic kit may be suitable for use in a cell-mediated immunity(CMI) assay. For example, the CMI assay may use detection of interferongamma (IFN-γ) as a readout system in either EIA (Wood & Jones, 2001) orELISPOT format (Vordermeier et al., 2002). As is well known to theskilled person, such assays do not depend on the detection of anantibody response but, instead, rely on recognition of an epitope by a Tcell, for example via binding of a T cell receptor.

In a further aspect of the present invention there is provided anisolated peptide of between 5 to 100 amino acids in length, for example8 to 100, 8 to 35, 8 to 25, 10 to 25 or 12-20 amino acids in length, inwhich the peptide has an epitope from M. bovis hypothetic proteinMb3645c (SEQ ID NO: 1) or from a polypeptide having at least 76%identity with SEQ ID NO: 1, and wherein the peptide has M. bovis- and/orM. tuberculosis-specific antigenic and/or immunogenic properties. Theisolated peptide may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length, for example 12or 20 amino acids in length.

The term “M. bovis—and/or M. tuberculosis—specific antigenic andimmunogenic properties” indicates that the peptide according to thisaspect of the invention is detectable by means of an immunogenic assay,preferably by means of a cell-mediated immunity (CMI) assay.

The isolated peptide may have an amino acid sequence of any of SEQ IDNOs: 2-13, or may comprise a contiguous combination of one or more ofthese sequences (for example, any two of SEQ ID NOs: 2-13 joinedtogether end-to-end).

The isolated peptide may be a peptide in which, compared with thecorresponding section of SEQ ID NO: 1, various amino acids have beendeleted. The peptide may thus be restricted to comprise the minimumnumber of amino acids required to maintain specificity against M. bovisand/or M. tuberculosis. For example, amino acid deletions may beacceptable provided that the tertirary structure of an epitope from SEQID NO:1 is maintained. A peptide modified in this way may be comprisedwithin a fusion peptide.

The present invention also encompasses variants of the diagnosticreagent peptide and the isolated peptide. As used herein, a “variant”means a peptide in which the amino acid sequence differs from the basesequence from which it is derived in that one or more amino acids withinthe sequence are substituted for other amino acids. The variant is afunctional variant, in that the functional characteristics of thepeptide from which the variant is derived are maintained. For example, asimilar immune response is elicited by exposure of an animal, or asample from an animal, to the variant polypeptide. In particular, anyamino acid substitutions, additions or deletions must not alter orsignificantly alter the tertiary structure of one or more epitopescontained within the peptide from which the variant is derived. Theskilled person is readily able to determine appropriate functionalvariants and to determine the tertiary structure of an epitope and anyalterations thereof, without the application of inventive skill.

Amino acid substitutions may be regarded as “conservative” where anamino acid is replaced with a different amino acid with broadly similarproperties. Non-conservative substitutions are where amino acids arereplaced with amino acids of a different type.

By “conservative substitution” is meant the substitution of an aminoacid by another amino acid of the same class, in which the classes aredefined as follows:

Class Amino acid examples Nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe,Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, GluBasic: Lys, Arg, His.

As is well known to those skilled in the art, altering the primarystructure of a peptide by a conservative substitution may notsignificantly alter the activity of that peptide because the side-chainof the amino acid which is inserted into the sequence may be able toform similar bonds and contacts as the side chain of the amino acidwhich has been substituted out. This is so even when the substitution isin a region which is critical in determining the peptide's conformation.

As mentioned above, non-conservative substitutions are possible providedthat these do not disrupt the tertiary structure of an epitope withinthe peptide, for example, which do not interrupt the immunogenicity (forexample, the antigenicity) of the peptide.

Broadly speaking, fewer non-conservative substitutions will be possiblewithout altering the biological activity of the polypeptide. Suitably,variants may be at least 50% identical, 60% identical, for example atleast 75% identical, such as at least 90% identical to the basesequence.

Also provided is an isolated nucleic acid which encodes a diagnosticreagent peptide, an isolated peptide, or variants thereof, as definedherein, but excluding the known ESAT-6 and CFP-10 polypeptides andpeptides defined by SEQ ID NO. 14-25. Using the standard genetic code, anucleic acid encoding an epitope or peptide may readily be conceived andmanufactured by the skilled person. The nucleic acid may be DNA or RNA,and where it is a DNA molecule, it may comprise a cDNA or genomic DNA.The invention encompasses fragments and variants of the isolated nucleicacid, where each such fragment or variant encodes a peptide withantigenic properties as defined herein. Fragments may suitably compriseat least 15, for example at least 30, or at least 60 consecutive basesfrom the basic sequence.

The term “variant” in relation to a nucleic acid sequences means anysubstitution of, variation of, modification of, replacement of deletionof, or addition of one or more nucleic acid(s) from or to apolynucleotide sequence providing the resultant peptide sequence encodedby the polynucleotide exhibits at least the same properties as thepeptide encoded by the basic sequence. In this context, the propertiesto be conserved are the ability to form one or more epitopes such thatan immune response is generated which is equivalent to that of thediagnostic reagent peptide or isolated peptide as defined herein. Theterm, therefore, includes allelic variants and also includes apolynucleotide which substantially hybridises to the polynucleotidesequence of the present invention. Such hybridisation may occur at orbetween low and high stringency conditions. In general terms, lowstringency conditions can be defined a hybridisation in which thewashing step takes place in a 0.330-0.825M NaCl buffer solution at atemperature of about 40-48° C. below the calculated or actual meltingtemperature (T_(m)) of the probe sequence (for example, about ambientlaboratory temperature to about 55° C.), while high stringencyconditions involve a wash in a 0.0165-0.0330M NaCl buffer solution at atemperature of about 5-10° C. below the calculated or actual T_(m) ofthe probe (for example, about 65° C.). The buffer solution may, forexample, be SSC buffer (0.15M NaCl and 0.015M tri-sodium citrate), withthe low stringency wash taking place in 3×SSC buffer and the highstringency wash taking place in 0.1×SSC buffer. Steps involved inhybridisation of nucleic acid sequences have been described for examplein Sambrook et al. (1989; Molecular Cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor).

Typically, variants have 60% or more of the nucleotides in common withthe nucleic acid sequence of the present invention, more typically 65%,70%, 80%, 85%, or even 90%, 95%, 98% or 99% or greater sequenceidentity.

Peptides and nucleic acids of the invention may be isolated from strainsof M. bovis and

M. tuberculosis. However, they may be prepared synthetically usingconventional peptide synthesisers. Alternatively, they may be producedusing recombinant DNA technology or isolated from natural sourcesfollowed by any chemical modification, if required. In these cases, anucleic acid encoding the peptide is incorporated into suitableexpression vector, which is then used to transform a suitable host cell,such as a prokaryotic cell such as E. coli. The transformed host cellsare cultured and the peptide isolated therefrom. Vectors, cells andmethods of this type form further aspects of the present invention.

In another aspect of the invention, there is provided a method fordiagnosing in a host an infection of, or exposure to, a mycobacterium,comprising the steps of:

i) contacting a population of cells from the host with a diagnosticreagent as defined herein; and

ii) determining whether the cells of said cell population recognise thediagnostic reagent.

The diagnostic reagent based on Mb3645c may be contacted together orseparately from the diagnostic reagent based on ESAT-6/CFP-10.

The population of cells may include T-cells. Recognition of thediagnostic reagent by said cells may be by way of, for example, bindingof a T cell receptor to the diagnostic reagent, for example, binding ofthe receptor to an epitope included within the diagnostic reagent. Themycobacterium may by M. bovis or M. tuberculosis.

The method for diagnosing may comprise a cell-mediated immunity (CMI)assay, for example a CMI assay which detects IFN-γ as described herein.

The term “polypeptide” as used herein includes long chain peptides, suchas proteins and epitopic fragments thereof. The term “peptide” refers tosmaller proteins, for example up to 100 amino acids in length.

Sequence identity between nucleotide and amino acid sequences can bedetermined by comparing an alignment of the sequences. When anequivalent position in the compared sequences is occupied by the sameamino acid or base, then the molecules are identical at that position.Scoring an alignment as a percentage of identity is a function of thenumber of identical amino acids or bases at positions shared by thecompared sequences. When comparing sequences, optimal alignments mayrequire gaps to be introduced into one or more of the sequences to takeinto consideration possible insertions and deletions in the sequences.Sequence comparison methods may employ gap penalties so that, for thesame number of identical molecules in sequences being compared, asequence alignment with as few gaps as possible, reflecting higherrelatedness between the two compared sequences, will achieve a higherscore than one with many gaps. Calculation of maximum percent identityinvolves the production of an optimal alignment, taking intoconsideration gap penalties.

Suitable computer programs for carrying out sequence comparisons arewidely available in the commercial and public sector. Examples includethe Gap program (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453)and the FASTA program (Altschul et al., 1990, J. Mol. Biol. 215:403-410). Gap and FASTA are available as part of the Accelrys GCGPackage Version 11.1 (Accelrys, Cambridge, UK), formerly known as theGCG Wisconsin Package. The FASTA program can alternatively be accessedpublically from the European Bioinformatics Institute and the Universityof Virginia. FASTA may be used to search a sequence database with agiven sequence or to compare two given sequences. Typically, defaultparameters set by the computer programs should be used when comparingsequences. The default parameters may change depending on the type andlength of sequences being compared. A sequence comparison using theFASTA program may use default parameters of Ktup=2, Scoringmatrix=Blosum50, gap=−10 and ext=−2.

BRIEF DESCRIPTION OF FIGURES

Particular non-limiting examples of the present invention will now bedescribed with reference to the following Figures, in which:

FIG. 1 is a histogram showing responder frequencies of screenedcandidate antigens;

FIG. 2 is a graph showing Mb3645c response in naïve cattle, infectedcattle, BCG vaccines and ESAT-6/CFP-10-negative samples. Recitation ofE6 and C10 are intended to refer to ESAT-6 and CFP-10, respectively;

FIG. 3 comprises graphs showing the correlation between mRNA abundanceand antigenicity in M. tuberculosis (left hand graph) and M. bovis(right hand graph);

FIG. 4 shows responses of peptides according to the invention,determined using an IFN-γ ELISPOT assay with PBMC isolated from M.bovis-infected cattle (sequence 02 corresponds to SEQ ID NO:2, sequence03 to SEQ ID NO:3 and so on). Numbers 3420, 3450 and 3606 areidentifiers for the three cattle tested; and

FIG. 5 shows a FACS analysis performed after stimulation of PBMCisolated from M. bovis-infected cattle with peptides according to theinvention (sequence 02 corresponds to SEQ ID NO:2, sequence 03 to SEQ IDNO:3 and so on, as above). Numbers 3420, 3450 and 3606 are identifiersfor the three cattle tested.

EXAMPLES Introduction

The identification of new subunit vaccine candidates or diagnosticmarkers has been greatly enhanced with the development of variouspost-genomic approaches (Cockle et al., 2002; Ewer et al., 2006). Thesehave largely involved sequence based analyses of the pathogen's genome.Here, the inventors took an alternative approach and focused on thetranscriptional activity of genes to identify potential antigens. Amethod of microarray analysis was developed that quantifies geneexpression on a global scale. As seen in Table 1, it was found that thatmany of the major mycobacterial antigens such as ESAT-6, CFP-10, Ag85Betc. are consistently highly expressed. Further to this, it was recentlyshown that the number of CD4+ T cells responsive to known mycobacterialantigens is closely related to the level of transcription of its gene(Rogerson et al., 2006).

With this as the basis, the inventors used a quantitative microarrayanalysis to identify genes that are consistently highly expressed inboth M. tuberculosis and M. bovis across a variety of growth conditions.Fourteen of these genes were then selected and screened for theirpotential as immunogens and diagnostic markers of infection using M.bovis infected cattle. No evidence was found to support a link betweenmRNA abundance and antigenicity. However, surprisingly, the inventorsstill identified one antigen that discriminated between infected andvaccinated cattle. Further, the same antigen showed a marked response ininfected cattle that do not respond to the classic mycobacterialantigens ESAT-6 and CFP-10, which will allow the antigen to increase thesensitivity of previously described differential diagnostic tests basedupon ESAT-6 and CFP-10 (Vordemeier et al. 2001).

Methods

Selection of Candidate Antigens

Six microarray datasets were used in this study. All RNA extraction andmicroarray hybridisations were performed as detailed in Bacon et al.(2004). The Perl computing language and the R statistical environmentwere used to perform all further data and statistical analysis.

For each data set, genome-wide mRNA abundances were calculated asfollows. Initially, all control spots on the array were removed from thedataset, including all representing ribosomal RNA. The local backgroundnoise, as determined by the image quantitation software, was subtractedfrom each spot. No data values were excluded from this study as it wasreasoned that weak signals (after background subtraction) werereflective of low abundance transcripts.

For each spot i on the array the fluorescent intensity from the RNAchannel was normalised by simple division to the fluorescent intensityof the gDNA channel:

Normalised Intensity (R _(i))=RNA_(i)/DNA_(i).

The correlation between hybridisation replicates within each dataset wasconfirmed to ensure there were no extreme outliers. Technical andbiological replicates were then averaged to provide a single normalisedintensity value for each gene on the array.

To account for an observed probe length bias, signal intensity wasnormalised to probe length using a model of linear regression of logintensity on probe length:

Probe normalised intensity (log^(e) Rn _(i))=log^(e) R_(i)−(intercept+slope*Probe Length_(i))

The corrected Rn_(i) values were converted back to a raw scale and forease of understanding are depicted as a proportional value, expressed inparts per million (ppm), based on the assumption that the sum of allintensity values represents the sum of the transcript (mRNA) populationwithin the sample:

ppm=(Rn _(i)/Σ_(i-ith) Rn)*10⁶

Candidate antigens were then selected based on their consistent highexpression across all six of the datasets which come from a variety ofexperimental conditions: M. tuberculosis in aerobic and low oxygenchemostats, M. tuberculosis in batch culture, M. tuberculosis inmacrophages, M. bovis in aerobic chemostats, and M. bovis in batchculture. Using these datasets, genes were selected which wereconsistently amongst the top 15% of abundant mRNA transcripts across allconditions in either M. tuberculosis, M. bovis or both. Candidates werefurther selected based on close amino acid homology between M.tuberculosis and M. bovis and little significant homology to otherclosely related species. Further to this, all candidates which had beentested previously were excluded. A total of 14 candidates were screenedusing 20mer overlapping peptides in this study (Table 1).

Cattle

10 uninfected control animals were obtained from herds within 4 yearlytesting parishes with no history of a BTB breakdown in the past 4 yearsand tested for the absence of an in vitro IFNγ response to PPD-A andPPD-B, to confirm an absence of infection. A further 20 animals fromsimilar BTB-free herds were vaccinated at least 6 months prior tosampling with 10⁶ CFU of BCG Danish strain 1331 (Statens SerumInstitute, Copenhagen, Denmark) according to the manufacturer'sinstructions (reconstituted in Sautons medium and 1 ml injectedsubcutaneously).

Blood samples were obtained from 30 naturally infected, tuberculin skintest positive reactors within herds known to have BTB. All animals wereadditionally screened for an in vitro IFNγ response to PPD-B and thepresence or absence of a response to ESAT-6 and CFP-10 was recorded.These animals were housed at VLA at the time of blood sampling.Infection was confirmed by necroscopy and/or M. bovis culture.

Production of Peptides

Bovine tuberculin (PPD-B) and avian tuberculin (PPD-A) were supplied bythe Tuberculin Production Unit at the Veterinary Laboratories Agency,Weybridge, Surrey UK and were used to stimulate whole blood at 10μg·ml⁻¹. Staphylococcal enterotoxin B was included as a positive controlat 5 μg·ml⁻¹.

Peptides representing our candidates were pin synthesised as 20-mersspanning the length of all 14 proteins with each peptide overlapping itsneighbour by 12 amino acid residues (Pepscan, Lelystad, Netherlands).These were dissolved in Hanks Balanced Salt Solution (Gibco) and 20%DMSO to 5 μg ml⁻¹ and grouped by gene into 26 pools of 8 to 12 peptides,with some genes represented by more than one pool. Pools were used tostimulate whole-blood at a final concentration of 10 μg·ml⁻¹ totalpeptide. Peptides from the ESAT-6 and CFP-10 proteins were synthesised,quality assessed and formulated into a peptide cocktail as previouslydescribed (Vordermeier et al., 2001).

IFN-γ/Enzyme-Linked Immunosorbent Assay

Whole-blood cultures were performed in 96-well plates where 250 μl wholeblood aliquots were mixed with antigen-containing solution to a finalconcentration of 10 μg·ml⁻¹. Serum containing supernatants wereharvested after 24 hours of culture at 37° C. and 5% CO₂ in a humidifiedincubator. The IFNγ concentration was determined using the BOVIGAM ELISAkit (Prionics AG, Switzerland). Results were deemed positive when theoptical densities at 450 nm (OD₄₅₀) with antigens minus the OD₄₅₀without antigens were ≧0.1. For comparative analysis of PPD-B versusPPD-A responses, a positive result was defined as a PPD-B OD₄₅₀ minusPPD-A OD₄₅₀ of ≧0.1 and a PPD-B OD₄₅₀ minus unstimulated OD₄₅₀ of >0.1.

BOVIGAM Data Analysis

All raw data from the BOVIGAM screening was handled using a PERLprogram, boviAnalyser.pl, which evoked analytical routines in thestatistical environment R (R-Development-Core-Team, 2006). Graphs weregenerated using both R and Graph-Pad Prism v4.

Ex Vivo IFN-γ Enzyme-Linked Immunospot (ELISPOT) Assay

Peripheral blood mononuclear cells (PBMC) were isolated from heparinizedblood taken from three cattle which had previously exhibited an in vitroresponse to the Rv3615c peptide pool. Separation was performed usingHistopaque 1077 (Sigma) gradient centrifugation, and the cells wereresuspended in RPMI 1640 tissue culture medium containing 25 mM HEPES(Gibco), 10% fetal calf serum, 1% nonessential amino acids, 5×10⁻⁵ Mβ-mercaptoethanol, 100 U/ml penicillin, and 100 μg ml⁻¹ streptomycin.Cells were enumerated, and suspensions containing 2×10⁶ cells ml⁻¹ wereprepared. IFN-γ production by PBMC was analyzed using a Mabtech bovineIFN-γ ELISPOT kit (Mabtech, Stockholm, Sweden). The ELISPOT plates(Multiscreen HTS-IP; Millipore) were coated at 4° C. overnight with abovine IFN-γ-specific monoclonal antibody, after which the wells wereblocked for 2 h using 10% fetal calf serum in RPMI 1640. The primaryantibody and blocking buffer were removed from the plates, and PBMCsuspended in tissue culture medium were then added (2×10⁵ cells well⁻¹)and incubated overnight at 37° C. with 5% CO2 in the presence of theindividual antigens. Stimulation was performed using the peptides at aconcentration of 5 μg ml⁻¹ or a pool of all 12 peptides containing 5 μgml⁻¹ of each peptide. The wells were washed using phosphatebufferedsaline plus 0.05% Tween 80. A secondary biotinylated antibody was usedat a concentration of 0.025 μg ml⁻¹ and this was followed by incubationwith streptavidin-linked horseradish peroxidase. After a further wash,the spotforming cells were visualized using an AEC chromogen kit(Sigma). Spots were counted using an AID ELISPOT reader and EliSpot 4.0software (Autoimmun Diagnostika, Germany).

Fluorescence-Assisted Cell Sorting (FACS) Analysis

PBMC were isolated from fresh heparinized blood as described above forthe ELISPOT assay and enumerated. Then a suspension containing 2×10⁶cells ml⁻¹ was prepared and incubated overnight in a 24-well plate(Nunc) at 37° C. in the presence of 5% CO2 with either RPMI medium(unstimulated control), PPD-B, pokeweed mitogen (positive control),individual peptides at a concentration of 5 μg ml⁻¹, or a pool of all 12peptides at a concentration of 5 μg ml⁻¹. After incubation, brefeldin A(Sigma) was added at a concentration of 10 μg ml⁻¹, and the preparationwas incubated for a further 4 h. The plate was centrifuged at 300×g for5 min, and the cells were resuspended in 250 μl (final volume) fortransfer to a 96-well plate. Surface antibody staining was performedusing Alexa Fluor 647-conjugated anti-CD4 (code MCA1653A627; Serotec)and fluorescein isothiocyanate-conjugated anti-CD8 (code MCA837F;Serotec) antibodies. Differential “live/dead” staining was performedusing Vivid (Invitrogen). After incubation for 15 min at 4° C., cellswere washed and centrifuged before they were permeabilized usingCytofix/Cytoperm (BD) at 4° C. for 20 min and stored overnight at 4° C.Intracellular staining for IFN-γ was performed usingR-phycoerythrin-conjugated anti-IFN-γ (Serotec) for 30 min at 4° C.Cells were finally suspended in 600 μl of buffer and analyzed using aCyan ADP instrument and the Summit 4.3 software (Dako, Denmark).

Results

Genes that had been found to be consistently highly expressed in M.tuberculosis and M. bovis across a variety of growth conditions (termedmembers of the abundant invariome) were assessed for the presence ofknown antigens. Ten previously well characterised antigens were found tobe a part of this abundant invariome (Table 1), which suggested thatother consistently highly expressed genes could also be antigenic.

TABLE 1 Mycobacterial antigens found to be highly expressed across avariety of growth conditions Rv Name Avg PPM StDev Reference Rv0288 cfp7781 286 (Skjot et al., 2002) Rv0440 groEL2 4438 2385 (Shinnick, 1987)Rv1174c Mpt8.4 1165 424 (Coler et al., 1998) Rv1886c fbpB/Ag85B 14641168 (Harth et al., 1996) Rv1987 Rv1987 495 136 (Cockle et al., 2002)Rv1980c mpt64 1316 629 (Harboe et al., 1986) Rv3418c groES 5189 2593(Baird et al., 1988) Rv3616c Rv3616c 2619 1457 (Mustafa et al., 2006)Rv3874 CFP-10 5414 3950 (Sorensen et al., 1995) Rv3875 ESAT-6 2472 1229(Berthet et al., 1998)

With this is mind, a list of 14 candidate antigens was generated basedon their consistent high expression across a variety of growthconditions. These included in vitro chemostat and batch cultures forboth M. tuberculosis and M. bovis, as well as for M. tuberculosisinfecting macrophages and growing in microaerophillic conditions. In themajority of cases, candidates were also selected based upon a closehomology between M. tuberculosis and M. bovis but with little homologyto other mycobacterial species (Table 2). The majority of the candidatesare annotated as conserved hypothetical proteins. However, three areputative membrane proteins, one is an excisionase and one a member ofthe PE family of proteins. Overlapping 20-mer peptides were synthesisedfor the complete coding sequence of each gene and were grouped into 26pools of 8 to 12 peptides, with some genes represented by more than onepool. These pools were then screened for their ability to stimulate anIFNγ response in vitro using whole blood from 30 M. bovis infected(bovine tuberculin (PPD-B) positive) and 10 M. bovis naive (PPD-Bnegative) cattle.

TABLE 2 Candidate antigens screened % aa seq homology to M. tuberculosis(“M. tb”) H37Rv if >50% Highly M. M. M. M. M. C. N. expressed Rv Mb M.tb bovis M. avium paratb leprae marinum smegmatis glutamicum farcinicain^(†): Function Rv1211 Mb1243 100 100 94 93 80 52 72 M. tb* CHP Rv1222Mb1254 100 100 64 64 71 67 64 Mb CHP Rv1398c Mb1433c 100 100 Mtb & MbCHP Rv2081c Mb2107c 100 100 Mb POSSIBLE TP Rv2876 Mb2901 100 99 68 58 50Mb POSSIBLE CONSERVED TP Rv3271c Mb3299c 100 100 78 Mtb PROB CONSERVEDIMP Rv3407 Mb3441 100 100 50 Mtb CHP Rv3477 Mb3504 100 98 70 70 58 75Mtb & Mb PE FAMILY PROTEIN (PE31) Rv3613c Mb3643c 100 100 M. tb* HPRv3614c Mb3644c 100 100 81 73 51 Mtb & Mb CHP Rv3615c Mb3645c 100 100 6779 Mtb & Mb CHP Rv3633 Mb3657 100 100 Mb CHP Rv3750c Mb3776c 100 100 MtbPOSSIBLE EXCISIONASE Rv3866 Mb3896 100 100 89 78 Mb CHP ^(†)Expressed inall 4 Mtb conditions (batch culture, aerobic and low oxygen chemostats,macrohpages) or 2 Mb conditions (batch and chemostat cultures) *In allMtb conditions except low oxygen CHP: Conserved Hypothetical Protein HP:Hypothetical Protein IMP: Integral Membrane Protein TP: TransmembraneProtein.

All M. bovis infected cattle had positive responses to PPD-B and inaddition 23 of the 30 infected cattle responded to an ESAT-6/CFP-10peptide cocktail (Vordermeier et al., 2001). The responder frequenciesfor all 14 candidate antigens in M. bovis infected and M. bovis naïvecattle are shown in FIG. 1. Seven of the candidates failed to stimulateany significant IFNγ response in either M. bovis infected or naïvecattle. Four of the candidate antigens stimulated a positive response in10% or more of the M. bovis naïve animals. This suggestedcross-reactivity with other environmental species even though theinventors had selected against significant homology in mycobacteriaother than M. tuberculosis or M. bovis. Four of the candidatesstimulated significant responses in M. bovis infected cattle, althoughtwo of these were recognised in 10% or less of the cattle tested and hadsimilar or greater responder frequencies in the PPD-B negative animals.Of the two remaining candidates Rv3750c/Mb3776c stimulated a response in15% of M. bovis infected cattle and none of the naïve animals.

Mb3645c was not recognised by any of the M. bovis naïve cattle, whereas11 of the 30 M. bovis infected animals (37%, p<0.01, FIG. 2) mounted apositive IFNγ response when stimulated with this peptide pool.Interestingly, given the recently proposed role for Rv3615c (the M.tuberculosis equivalent to Mb3645c) in the secretion of ESAT-6/CFP-10 inM. tuberculosis (Macgum et al., 2005, Fortune et al., 2005), it wasnoted that positive responses to the Mb3645c peptide pool from M. bovisinfected animals were enriched in cattle that did not respond to ESAT-6or CFP-10 (4 of 7, 57%, p<0.05, FIG. 2). This raises the possibilitythat these proteins could be used to increase the sensitivity ofpreviously developed ESAT-6/CFP-10 based diagnostic tests (Vordermeieret al., 2001).

To assess Mb3645c's potential as an antigen for differential diagnosisof BCG vaccinated and M. bovis infected animals, the peptide pool in 20BCG vaccinated cattle was screened. In contrast to M. bovis infectedanimals, none of the BCG vaccinated cattle generated a significant IFNγresponse to the Mb3645c peptides (p<0.01, FIG. 2).

Finally, as few of the candidates turned out to be potent antigens, thecorrelation between mRNA levels and antigenicity was further explored.The responder frequencies were collected for an additional 80mycobacterial proteins that had been screened in M. bovis infectedcattle (Ewer et al., 2006, Cockle et al., 2002, Mustafa et al., 2006).Together these 94 proteins had responder frequencies that varied from 0to 86% with an average of 30% so represented a broad range of antigenicpotential. In comparison to their mRNA abundances, little correlationwas found in either chemostat grown M. tuberculosis or M. bovis: 0.01(Spearman's, p=0.38) and 0.06 (Spearman's, p=0.56) respectively,suggesting that mRNA level alone is not a strong predictor for antigenicpotential in cattle.

To confirm the presence and location of the T-cell epitopes withinRv3615c, the response to constituent peptides from the Rv3615c pool wasdetermined using an IFN-γ ELISPOT assay with PBMC isolated from M.bovis-infected cattle. Peptides SEQ ID NOs: 8-13 were recognized in atleast two of the three cattle tested. Peptides SEQ ID NOs: 10-13(spanning amino acids 57 to 103) from the C terminus of the protein werethe most antigenic and were recognized by all three animals tested.Peptide SEQ ID NO:13 (AKIYSEADEAWRKAIDGLFT), in particular, stimulated aresponse in all three animals, with an average of 509 spot-forming units(SFU) per 10⁶ PBMC (standard deviation, 185.3 SFU per 10⁶ PBMC), whichis comparable to the results for the pool as a whole (414 SFU per 10⁶PBMC; standard deviation, 135.6 SFU per 10⁶ PBMC) (FIG. 4).

To further characterize the specific lymphocyte response to Rv3615c, aFACS analysis was performed with PBMC isolated from the same M.bovis-infected cattle that were used for the ELISPOT analysis.Lymphocytes were analyzed for intracellular IFN-γ production and thepresence of CD4 and CD8 cell differentiation markers. It was found that,mirroring the ELISPOT data, peptides SEQ ID NO:2-8 stimulated littleIFN-γ production. Markedly higher levels of IFN-γ were observed for thecells stimulated with peptides SEQ ID NOs: 9, 10, 12 and 13 (FIG. 5).Interestingly, no IFN-γ response to peptide SEQ ID NO: 11 was observed,despite the fact that a response was recorded in the ELISPOT assay.Analysis of the cells stimulated with peptide SEQ ID NO: 11 showed thatthe majority (>64%) of the cells in the sample were dead, suggestingthat the peptide itself caused IFN-γ-induced apoptosis, which would bein line with the positive responses seen in the ELISPOT assay.

DISCUSSION

There have been many strategies for the identification of mycobacterialimmunogens using post-genomic methods including T-cell epitopeprediction (Vordermeier et al., 2003) and genomic comparisons toidentify pathogen specific open reading frames (Ewer et al., 2006).Previous work had shown that many highly expressed genes were knownmycobacterial antigens; therefore, the inventors consideredtranscriptional activity as a predictor of antigenicity.

A selection of consistently highly expressed genes were screened fortheir ability to stimulate IFNγ responses from cattle infected with M.bovis. Fourteen candidates were selected based on their high expressionin both M. tuberculosis and M. bovis across a variety of growthconditions, including in vitro chemostat and batch cultures, as well asfrom a macrophage infection and microaerophillic chemostat cultures.Proteins were excluded if they were known immunogens or had significanthomology to proteins in other mycobacteria; hence the majority of thecandidate antigens had no functional annotation. However, three werepredicted membrane associated proteins, one an excisionase and one a PEfamily protein (Table 2).

Three of the candidates screened here (Rv3615c/14c/13c) appear to belocated in the same operon of five genes (Rv3616c to Rv3612c). Theentire operon is consistently highly expressed across all of the growthconditions analysed by microarray. One of these candidates—Mb3645c—hadthe greatest responder frequency in M. bovis-infected cattle of all ofthe candidates tested in this study. The products of these operonicgenes have been identified as components of the mycobacterial secretionsystem (the SNM system), which functions to export both ESAT-6 andCFP-10 (Macgurn et al., 2005, Fortune et al., 2005). The product of thefirst gene in this operon, Rv3616c, has also been shown to be a dominantmycobacterial antigen. Rv3616c is more frequently recognised in M. bovisinfected cattle compared to Rv3615c: 84.6% versus 37% (Mustafa et al.,2006). Rv3616c is secreted in a mutually dependent manner with ESAT-6and CFP-10 (Fortune et al., 2005), whereas Rv3615c appears to interactwith other proteins of the secretion system (Macgurn et al., 2005) andmay therefore remain within the bacterial cell, which could explain thedifference in frequencies of response from M. bovis infected cattle.

The ESAT-6/CFP-10 peptide cocktail had been developed as an alternativediagnostic reagent to PPD and differentiates infected and vaccinatedindividuals as these antigens are not present in M. bovis BCG(Vordermeier et al., 2001). The test is reported to have a sensitivityof around 77.9% in infected cattle. Rv3615c has been found not to berecognised by the immune systems of either M. bovis naïve or BCGvaccinated animals, unlike Rv3616c to which 40% of vaccinatedindividuals respond (Mustafa et al., 2006), and is therefore highlyspecific. Furthermore, 57% of cattle infected with M. bovis which do notrespond to the ESAT-6/CFP-10 peptide cocktail used did generate asignificant IFNγ response to Rv3615c. Therefore, the inclusion ofRv3615c into the ESAT-6/CFP-10 diagnostic cocktail increases thesensitivity of a diagnostic test for M. bovis, by detecting infectedanimals that fail to recognise the ESAT-6/CFP-10 epitopes. This isachieved without compromising test specificity.

Of the initial 14 candidates, just one was significantly antigenicwhereas previous observations had led workers to believe that manyhighly expressed genes could be potent immunogens. It is well known thatthe processes of transcription and translation are tightly coupled inprokaryotes (Miller et al., 1970) and some correlation between mRNA andprotein levels exists in the mycobacteria. It was therefore hypothesisedthat this was potentially reflecting a trend whereby more abundantproteins are simply more accessible to the host immune systems and morelikely to be processed and presented by phagocytes. In light of the datagenerated by the inventors, this was examined in more detail bycollecting responder frequencies for 94 proteins, including the 14screened in this study. Surprisingly, very little correlation was foundbetween mRNA levels and antigenicity as measured by responderfrequencies, suggesting that mRNA abundance alone is not a validpredictor of antigenic status.

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Although the present invention has been described with reference topreferred or exemplary embodiments, those skilled in the art willrecognize that various modifications and variations to the same can beaccomplished without departing from the spirit and scope of the presentinvention and that such modifications are clearly contemplated herein.No limitation with respect to the specific embodiments disclosed hereinand set forth in the appended claims is intended nor should any beinferred.

All documents cited herein, along with priority application Nos.: U.S.Ser. No. 15/016,859, U.S. Ser. No. 14/186,291, U.S. Ser. No. 12/742,223,PCT/GB2008/003724, and GB 0722105.4, including Sequence Listings, areincorporated by reference in their entirety.

1: A diagnostic reagent for use in the detection of M. tuberculosisinfection in a human, comprising a peptide having an amino acid sequenceat least 90% identical to SEQ ID NO: 1, said peptide comprising anepitope consisting of amino acids from within SEQ ID NO: 1, wherein saidpeptide has fewer than 103 amino acids. 2: The diagnostic reagentaccording to claim 1, wherein said epitope is a T-cell epitope. 3: Thediagnostic reagent according to claim 1, wherein said diagnostic reagentis a cell-mediated immunity (CMI) assay diagnostic reagent. 4: Thediagnostic reagent according to claim 1, wherein said epitope is aT-cell epitope, and wherein said diagnostic reagent is a cell-mediatedimmunity (CMI) assay diagnostic reagent. 5: The diagnostic reagentaccording to claim 1, wherein said peptide is a truncated form of SEQ IDNO:
 1. 6: The diagnostic reagent according to claim 1, wherein saidepitope has 5 or more amino acids. 7: The diagnostic reagent accordingto claim 1, further comprising one or more peptides each selected fromamino acid sequences SEQ ID NO:14-25. 8: The diagnostic reagentaccording to claim 1, further comprising one or more peptides eachselected from amino acid sequences SEQ ID NO:16-25. 9: The diagnosticreagent according to claim 1, further comprising one or more peptideseach selected from amino acid sequences SEQ ID NO:14-25 and one or morepeptides each selected from amino acid sequences SEQ ID NO:16-25. 10: Adiagnostic reagent for use in the detection of M. tuberculosis infectionin a human, comprising a peptide having an amino acid sequence at least90% identical to SEQ ID NO: 1, said peptide comprising an epitopeconsisting of amino acids from within SEQ ID NO: 1, wherein said peptidehas 100 amino acids or fewer. 11: The diagnostic reagent according toclaim 10, wherein said peptide has 75 amino acids or fewer. 12: Thediagnostic reagent according to claim 11, wherein said peptide has 50amino acids or fewer. 13: The diagnostic reagent according to claim 12,wherein said peptide has 25 amino acids or fewer. 14: The diagnosticreagent according to claim 13, wherein said peptide has 20 amino acidsor fewer. 15: The diagnostic reagent according to claim 10, wherein saidepitope is a T-cell epitope, and wherein said diagnostic reagent is acell-mediated immunity (CMI) assay diagnostic reagent. 16: Thediagnostic reagent according to claim 10, wherein said epitope has 5 ormore amino acids. 17: The diagnostic reagent according to claim 10,further comprising one or more peptides each selected from amino acidsequences SEQ ID NO:14-25 or one or more peptides each selected fromamino acid sequences SEQ ID NO:16-25. 18: The diagnostic reagentaccording to claim 17, further comprising one or more peptides eachselected from amino acid sequences SEQ ID NO:14-25 and one or morepeptides each selected from amino acid sequences SEQ ID NO:16-25. 19: Amethod for diagnosing, in a human, infection by or exposure to M.tuberculosis versus prior M. bovis bacillus Calmette-Guerin (BCG)vaccination, comprising the steps of: (a) contacting a population ofcells from said human with a diagnostic reagent comprising a peptidehaving an amino acid sequence at least 90% identical to SEQ ID NO: 1,said peptide comprising an epitope consisting of amino acids from withinSEQ ID NO: 1; and (b) determining whether cells of said population ofcells recognize the epitope; wherein recognition by said population ofcells of said epitope indicates infection by or exposure to M.tuberculosis irrespective of prior M. bovis bacillus Calmette-Guerin(BCG) vaccination. 20: The method according to claim 19, whereindetermining step (b) comprising comparing IFNγ levels to a control. 21:A method of diagnosing M. tuberculosis infection in a human, or ofdetermining whether a human has been exposed to M. tuberculosis,comprising: (i) contacting T cells from said human with one or more of(a) a peptide having the sequence shown in SEQ ID NO: 1 (b) a peptidehaving a sequence of at least 8 consecutive amino acids of the sequenceshown in SEQ ID NO: 1; or (c) a peptide having a sequence that iscapable of binding to a T-cell receptor which recognizes a peptidehaving the sequence shown in SEQ ID NO: 1 or a sequence of at least 8consecutive amino acids of the sequence shown in SEQ ID NO: 1; and (ii)determining whether the said T-cells recognize said peptide, whereinrecognition of said peptide by said T-cells is determined by detecting acytokine from the T cells using a cell-mediated immunity (CMI) assay.22: The method of claim 21, wherein step (i) further comprisescontacting said T cells from said human with one or more M. tuberculosisantigens. 23: The method of claim 22, wherein said one or more M.tuberculosis antigens include antigens encoded by the RD-1 region, whichantigens are selected from ESAT-6, CFP10, fragments thereof with SEQ IDNOs: 16-25, or combinations thereof. 24: The method of claim 21, whereinone or more of the peptides: (i) is represented by SEQ ID Nos: 2-13, or(ii) binds to a T-cell that recognizes (i), are contacted with theT-cells. 25: The method of claim 21, wherein said cytokine is IFN-γ. 26:The method of claim 21, wherein said cytokine is detected by anenzyme-linked immunosorbent assay. 27: The method of claim 21, whereinsaid T-cells have been cultured in vitro. 28: The method of claim 21,wherein said peptide has fewer than 103 amino acids. 29: The method ofclaim 21, wherein said peptide has up to 100 amino acids. 30: A kit fordiagnosing M. tuberculosis infection or exposure in a human, said kitcomprising a composition comprising: (a) a peptide having the sequenceshown in SEQ ID NO: 1; (b) a peptide having a sequence of at least 8consecutive amino acids of the sequence shown in SEQ ID NO: 1; (c) apeptide having a sequence which is capable of binding to a T-cellreceptor which recognizes a peptide defined in (a) or (b); or (d) atleast one peptide: (i) which is presented by SEQ ID Nos: 2-13, or (ii)which binds to a T-cell that recognizes (i), and the compositionoptionally comprises one or more further M. tuberculosis T-cellantigens, wherein the kit comprises a means for detecting recognition ofa peptide by a T-cell. 31: The kit of claim 30, wherein said one or morefurther M. tuberculosis T-cell antigens is selected from ESAT-6 and/orCFP10, fragments with SEQ ID NOs: 16-25 or combinations thereof. 32: Thekit of claim 30, wherein said means for detecting recognition of saidpeptide by T-cells is a cell-mediated immunity (CMI) assay. 33: The kitof claim 30, wherein said peptide has fewer than 103 amino acids. 34:The kit of claim 30, wherein said peptide has up to 100 amino acids.